Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells
Fig 3
Loss of CypA-DsRed following virus-cell fusion.
VSV-G pseudotyped HIV-1 particles bearing WT CA or CA mutants and co-labeled with IN-sfGFP and CypA-DsRed were used to infect TZM-bl cells (~10 pg of p24 per 5·104 cells, MOI 0.008). Viruses were pre-bound to cells in the cold and virus entry/fusion was initiated by adding a pre-warmed buffer. Time-lapse images of a single field of view were taken every 30 sec for 80 min, at which point, 10 μM CsA was added and image acquisition continued for 40 min. (A) Kinetics of CypA-DsRed loss from IN-sfGFP puncta was measured for WT CA and each of the CA mutant viruses in 5 independent experiments. Inset: The fraction of double-labeled cores that lose CypA-DsRed in response to 10 μM CsA addition at 80 min (arrow). (B) Same as in panel A, but CsA was added just before starting the image acquisition (0 min) to measure the fusion kinetics. (C, D) Selected images and corresponding CypA-DsRed and IN-sfGFP intensity profiles obtained by single particle tracking. The tracks and images illustrate the relatively quick (C) and the less frequently occurring slow (D) release of CypA-DsRed. Scale bar 0.5 μm. (E) Effects of CA-binding compounds PF74 (10 μM) and BI-2 (20 μM) on the kinetics of CypA-DsRed loss from single post-fusion cores. (F) Reverse transcription accelerates the loss of CypA-DsRed from IN-sfGFP labeled particles. Inhibition of viral DNA synthesis by Nevirapine (10 μM) or by the RT D185N mutation, delayed the kinetics of CypA-DsRed disappearance after fusion. The E478Q mutation, which abolishes RNase H activity, did not exert a significant effect on the CypA-DsRed retention time. Insets to panels E and F: The fraction of double-labeled cores that lose CypA-DsRed in response to 10 μM CsA added at 80 min (arrows). Statistical significance was determined by the Mann-Whitney Rank-Sum test.